1. Field of the Invention
The present invention relates to methods of reducing substitution of amino acids during the production of polypeptides of interest in mammalian cells. By varying the concentration of amino acids in the culture medium, heterogeneity of the polypeptide's amino acid sequence is decreased, leading to higher yields of protein of desired sequence.
2. Background
Substitutions of amino acids in the primary sequence of a protein are known to occur in nature. Typically, mutation occurs at the DNA level and then gets translated to the protein. During recombinant protein production, variant sequences including point mutations have been known to be introduced into production cell lines. For example, a variant form of an antibody gene sequence was found to be developed during the transfection of antibody light and heavy chain genes into chinese hamster ovary (CHO) cells (Harris, R. J. et al. Bio-Technology 11:1293-1297 (1993)).
Substitution can also occur during protein synthesis. Substitution during protein synthesis has been shown in ribosomal and non-ribosomal synthesis of peptides and proteins in prokaryotic systems. Mistranslations can be result of aberrant initiation, a frame shift, a missense error, tRNA hopping, or a termination bypass (Santos and Tuite, Trends Biotech. 11:500-505 (1993)). Imbalances in translation machinery typically impact the expression levels at which the protein of interest is expressed. In extreme cases of imbalance, increased protein heterogeneity due to misincorporation of amino acids has been observed.
The present invention reports on recombinant expression of polypeptides of interest in mammalian cells wherein a depletion of particular amino acids during synthesis triggers misincorporation.